Gardimycin and process for producing same

ABSTRACT

The antibiotic gardimycin and process for producing said antibiotic by fermentation of strains belonging to the genus Actinoplanes.

The present invention refers to the production and the isolation of a new family of antibiotic substances obtained by fermentation of strains belonging to the genus Actinoplanes. These substances will hereafter be referred to as metabolite A, metabolite B and metabolite C: metabolite B is also named gardimycin.

As stated above the antibiotic substances are produced by cultivation of fermenting strains belonging to the genus Actinoplanes. These strains were isolated from soil samples collected at the localities Temossi (Italy) and Garbadi Bridge (India). Our collection numbers are A/6353 for the strain isolated from the Italian soil sample and A/10889 for the strain isolated from the Indian soil sample: both have been deposited and made part of the stock culture collection of ATCC where they were assigned the numbers 31048 and 31049 respectively.

In the preparation of the new antibiotic substances the selected organism is cultivated under aerobic conditions in an aqueous nutrient medium containing a source of carbon, a source of nitrogen and inorganic salts. Ordinarily the strain is precultured in a shake flask, until substantial antibiotic activity is present, then the culture is used to inoculate jar fermentors containing a nutrient fermentative medium. Cultivation is continued at 25°-35° C. under aerobic conditions for a time sufficient to produce a substantial antibiotic level. During this time microbiological assays are carried out by the agar diffusion method to control the concentration of the antibiotic substance produced. Sarcina lutea is used as the test organism.

The so obtained antibiotic activity can be isolated from the fermentation broth by conventional procedures, such as, for instance, by extraction with an organic solvent in which the antibiotic activity is soluble and which is immiscible with the aqueous medium. For this purpose, organic solvents selected from lower alkanols of 3 to 6 carbon atoms and (C₁₋₄)-lower halogenated hydrocarbons may advantageously be employed.

The organic phase is separated from the aqueous medium, concentrated to small volume and allowed to stand for 10-15 hours at low temperature until a precipitate forms, which is recovered by filtration. Paper chromatography on Whatman paper No. 1 and thin layer chromatography on silicagel of the obtained crude product, and subsequent microbiological development (Nicolaus et al., Il Farmaco, Ed. Prat., 8, 350-370, 1961) on Staphylococcus aureus as the detecting system, indicate the presence of at least two active components, which for identification purposes, are named metabolite A and metabolite B, the latter being produced in higher amounts than the former. These two components have different R_(f) values which vary depending on the nature of the eluting system. Metabolites A and B may be purified and isolated as the pure compounds by means of usual techniques, such as, for instance, by several countercurrent extractions in a predetermined solvent system in which the two antibiotic substances have different partition coefficients. During this step metabolite B is recovered as the monosodium salt, which can in turn be transformed into other salts with alkali or alkali earth metals, or into the corresponding free acid, by means of common operations.

The mother liquors deriving from the butanol extract are poured into an inert, non-polar organic solvent, as, for instance, light petroleum, and a further precipitate forms.

Chromatographic investigations and microbiological developments carried out as above indicate that the obtained solid essentially consists of a single product, which is different from metabolites A and B owing to the different R_(f) values in the same eluting system: for identification purposes it is named metabolite C. The chromatographic patterns of the three metabolites A, B and C will be hereinafter reported.

Metabolite A, metabolite B (hereafter referred to as gardimycin) as well as its salts with alkali and alkali earth metals and metabolite C show good antibacterial in vitro and in vivo activities. More particularly, gardimycin exhibits an outstanding in vitro antimicrobial action especially against gram-positive bacteria an concentration levels between 1 and 50 μg/ml, as it results from the following table, reporting the minimal concentration of antibiotic substance which inhibits the growth of various pathogenic microorganisms.

                  TABLE 1                                                          ______________________________________                                                              Minimal inhibitory                                               Strain        concentration (μg/ml)                                  ______________________________________                                         Staphylococcus aureus ATCC 538                                                                      50                                                        Micrococcus flavus ATCC 10240                                                                       1                                                         Streptococcus faecalis ATCC 10541                                                                   50                                                        Streptococcus haemolyticus C 203                                                                    2                                                         Diplococcus pneumoniae UC 41                                                                        50                                                        Clostridium perfringens ISS 30543                                                                   2                                                         Neisseria gonorrheae ATCC 9826                                                                      20                                                        ______________________________________                                    

Another favorable characteristic of the antibiotic substance gardimycin is that it is active against clinically isolated Streptococcus strains.

                  TABLE 2                                                          ______________________________________                                                              Minimal inhibitory                                        Strain               concentration (μg/ml)                                  ______________________________________                                         Streptococcus haemolyticus                                                                     2078     1                                                     Streptococcus haemolyticus                                                                     2087     1                                                     Streptococcus viridans                                                                         2057     5                                                     Streptococuss viridans                                                                         2085     2                                                     ______________________________________                                    

Furthermore, as stated above, gardimycin displays also a very interesting in vivo activity against experimental infections in mice caused by pathogenic bacteria of the genus Diplococcus and Streptococcus. The hereinbelow reported table shows the ED₅₀ values of gardimycin against experimental infections provoked by Streptococcus haemolyticus C 203 and Diplococcus pneumoniae UC 41.

                  Table 3                                                          ______________________________________                                         Strain                 ED.sub.50 mg/Kg                                         ______________________________________                                         Streptococcus haemolyticus C 203                                                                      0.75                                                    Diplococcus pneumoniae UC 41                                                                          30                                                      ______________________________________                                    

These favorable antimicrobial properties are coupled with a very low toxicity as the LD₅₀ value of the antibiotic gardimycin is higher than 1000 mg/Kg i.p. and about 2000 mg/Kg i.v.

DESCRIPTION OF THE PRODUCING STRAINS Description of strain A/6353

The strain grows well on several agar media. On oat meal agar the colonies, which are of about 5 mm. in diameter, have indented contours, slight radial furrows and a central depression. Aerial mycelium is always absent. Sporangia form abundantly on oat meal agar, glycerol asparagine agar and Czapek glucose agar, showing different shape and size depending on the medium.

On oat meal agar they have regular contours and a shape varying from spherical to oval. The sporangia size is in the range 15-25 μ. The spores are motile and spherical with a diameter of 1.5-2 μ. A yellow-amber soluble pigment is produced in several media.

Description of Actinoplanes A/10889

The strain grows well on various nutrient agars. The surface is opaque and usually rough to wrinkled. Aerial mycelium is usually absent, however in some media rudiments of aerial mycelium are observed. At the microscopic examination the vegetative mycelium is slightly branched with a diameter of ˜1μ. The sporangia form moderately only on calcium malate agar and are globose with irregular surface often lobate, with diameter ranging from 7.0 to 12.0μ. After rupture of the sporangial wall it is possible to observe sporangial release. The subspherical spores are motile (1.0 to 1.5μ diameter).

A comparison of some morphological characteristics of the two strains is shown in table 4.

                  Table 4                                                          ______________________________________                                                   Strains                                                                           A/10889      A/6353                                               ______________________________________                                         Sporangia   formed only on Ca                                                                            formed abundantly on                                             malate agar   several agars; but                                               Size: 7×12 mμ                                                                       variable in size and                                                           shape. On oat meal                                                             agar the size is                                                               15-25 mμ                                          Spores      subspherical  spherical                                                        (1 to 1.5 μ m)                                                                            (1.5-2 mμ)                                        Aerial mycelium                                                                            rudimentary   always absent                                        Soluble pigment                                                                            absent        yellow-amber                                                                   pigment present on                                                             some agars                                           ______________________________________                                    

Table 5 reports the cultural characteristics of Actinoplanes A/6353 and Actinoplanes A/10889 cultivated on various standard media suggested by Shirling and Gottlieb (Intern.J.Syst.Bact. 16, 313-340, 1966) and other media recommended by Waksman (The Actinomycetes, vol. II, The Williams and Wilkins Co., 1961). The cultural characteristics were determined after 6 to 14 days of incubation at 30° C.

                                      Table 5                                      __________________________________________________________________________     The number of some of the culture media refers to those given by Shirling      and Gottlieb                                                                   __________________________________________________________________________                                Cultural Characteristics                            Culture Medium             A/10889               A/6353                        __________________________________________________________________________     Medium n. 2        Abundant growth, wrinkled surface,                                                                  Abundant growth, slightly                                                      wrinkled                               (Yeast exctract-malt agar)                                                                        amber                light orange to light amber            Medium n. 3        Moderate growth, smooth, opaque,                                                                    Abundant growth, with smooth and                                               thin                                   (Oatmeal agar)     cream to orange at edges                                                                            surface, light orange. Some                                                    sporangia, hight yellow soluble                                                pigment                                Medium n. 4        Moderate growth, smooth surface, deep                                                               Abundant growth with smooth                                                    surface                                (Inorganic salts-starch agar)                                                                     orange               orange. Some sporangia. Canary                                                 yellow soluble pigment                 Medium n. 5        Moderate growth, smooth surface,                                                                    Abundant growth with smooth                                                    surface,                               (Glycerol-asparagine agar)                                                                        orange               light orange. Abundant                                                         production                                                                     of pigment. Canary yellow                                                      soluble                                                                        pigment                                Medium n. 6        Scanty growth, rough surface, dark                                                                  Moderate growth with smooth                                                    surface                                (Peptone-yeast exctract-iron agar)                                                                brown, with brown pigment                                                                           orange                                 Medium n. 7        Abundant growth, crusty surface,                                                                    Abundant growth with smooth                                                    surface,                               (Tyrosine agar)    coffee colored       rose amber. Good production of                            Weakly brown pigment sporangia. Rose amber soluble                                                  pigment                                Oatmeal agar (according to                                                                        Abundant growth, crusty surface,                                                                    Abundant growth, with smooth                                                   surface,                               Waksman)           orange               light orange to topaze yellow                             Traces of rudimentary aerial mycelium                                                               Good production of big                                                         sporangia,                                                                     yellow soluble pigment                 Hickey and Tresner's agar                                                                         Abundant growth, wrinkled surface,                                                                  Abundant growth with smooth                                                    surface rose                                              light brown          amber                                                                          Abundant production of sporangia       Czapek glucose agar                                                                               Poor growth, smooth surface, light                                                                  Abundant growth with smooth and                                                thin                                                      orange               surface, orange                                                                Abundant production of sporangia                          Traces of rudimentary aerial mycelium                       Glucose asparagine agar                                                                           Abundant growth, crusty surface, deep                                                               Abundant growth with smooth                                                    surface                                                   orange               orange.                                                                        Some sporangia                         Nutrient agar      Moderate growth, crusty surface,                                                                    Abundant growth with smooth                                                    surface,                                                  orange to light brown                                                                               orange                                 Potato agar        Abundant growth, wrinkled surface,                                                                  Abundant growth, with smooth                                                   surface                                                   orange to light brown.                                                                              amber                                                     Traces of rudimentary aerial mycelium                       Bennett's agar     Abundant growth, wrinkled surface,                                                                  Abundant growth with crusty                                                    surface                                                   light orange.        light orange                           Calcium malate agar                                                                               Moderate growth, crusty surface,                                                                    Scarce growth with smooth                                                      surface.                                                  cream to light orange.                                                                              colorless. Some sporangia              Skim milk agar     Abundant growth, wrinkled surface,                                                                  Abundant growth with slightly                                                  crusty                                                    deep orange.         surface, deep orange                                                           yellow soluble pigment                 Czapek agar        Poor growth, crusty surface, light                                                                  Scarce growth light orange                                orange               Moderate production of sporangia       Egg agar           Abundant growth, smooth surface,                                                                    Abundant growth, with smooth                                                   surface,                                                  orange               light creme. Moderate production                                               of sporangia                           Peptone glucose agar                                                                              Very scant growth, smooth surface,                                                                  Abundant growth, with wrinkled                                                 surface,                                                  hyaline              deep orange                            Agar               Very scanty growth, thin and smooth                                                                 Very scanty growth thin and                                                    smooth.                                                   hyaline              Colorless                              Loeffler serum     Very scanty growth, smooth surface,                                                                 Scarce growth, light orange                               light orange to amber                                       Potato             Scanty growth, wrinkled surface,                                                                    Scarce growth, wrinkled, light                                                 orange                                                    orange                                                      __________________________________________________________________________

The most convenient temperature for development of the colonies was found to range from about 18° to about 42° C., the optimum temperature being from about 28° C. to about 37° C.

Table 6 reports the utilisation of carbon sources examined according to the method of Pridham and Gottlieb.

                  Table 6                                                          ______________________________________                                         Carbon Utilization                                                             ______________________________________                                                         Growth                                                         Carbon source     A/10889     A/6353                                           ______________________________________                                         Inositol          -           +                                                Fructose          +           +                                                Rhamnose          +           +                                                Mannitol          +           -                                                Xylose            +           +                                                Raffinose         -           -                                                Arabinose         +           +                                                Cellulose         -           -                                                Salicin           +           -                                                Sucrose           +           -                                                Mannose           +           +                                                Lactose           +           -                                                Glucose (positive control)                                                                       +           +                                                ______________________________________                                    

Table 7 reports physiological characteristics of the two strains.

                  Table 7                                                          ______________________________________                                         Physiological characteristics                                                  ______________________________________                                                Test       A/10889     A/6353                                           ______________________________________                                         Starch hydrolysis positive    positive                                         H.sub.2 S formation                                                                              positive    negative                                         Melanin production                                                                               positive    negative                                         Tyrosine hydrolysis                                                                              positive    negative                                         Casein hydrolysis negative    positive                                         Ca-malate hydrolysis                                                                             negative    negative                                         Litmus milk coagulation                                                                          negative    negative                                         Litmus milk peptonization                                                                        negative    negative                                         Nitrate reduction positive    negative                                         Gelatin liquefaction                                                                             positive    negative                                         ______________________________________                                    

The two strains A/6353 and A/10889 because of their global sporangia, motile spores and colony morphology are ascribed to the genus Actinoplanes. However, they are clearly different from each other on the basis of their growth pattern on different agars, production or non-production of soluble pigment, carbon utilization pattern, and physiological characteristics. In particular, A/6353 shows a very limited ability to hydrolize glycosidic bonds, including sucrose which is utilized by all the species of Actinoplanes so far described. A/10889 produces a rudimentary aerial mycelium, seldom found in Actinoplanes species. The two strains are also easily distinguishable from all the Actinoplanes species so far known. For these reasons, strain A/6353 and strain A/10889 are recognized as new species of Actinoplanes and are given the names Actinoplanes liguriae ATCC 31048 and Actinoplanes garbadinensis ATCC 31049.

EXAMPLE 1 Production of the antibiotic, isolation and separation of the various metabolities

For producing the antibiotic activities the strain Actinoplanes Garbadinensis ATCC 31049 is aerobically pre-cultured in a nutrient medium until substantial antibiotic activity is present. As an example a shake flask culture may have the following composition in g/l.

    ______________________________________                                         Meat extract           3.0                                                     Yeast extract          10.0                                                    Calcium carbonate      4.0                                                     Starch                 25.0                                                    Tap water q.s. to 1000 ml.                                                     ______________________________________                                    

The flasks are shaken for about 24 hours at about 28°-30° C. and then the pre-cultures (one liter) are used to inoculate jar fermentors each containing 10 liters of the following nutrient medium:

    ______________________________________                                         Meat extract         40          g                                             Peptone              40          g                                             Yeast extract        10          g                                             Sodium chloride      25          g                                             Soybean meal         100         g                                             Glucose              500         g                                             Calcium carbonate    50          g                                             Tap. water q.s. to 10 liters                                                   ______________________________________                                    

The fermentation batches are incubated aerobically under stirring at 28°-30° C. At intervals the antibiotic activity is assayed microbiologically by the agar diffusion method using Sarcina lutea as the test organism. The maximum activity is reached after 96-120 hours of fermentation.

The fermentation broth is adjusted at pH 8.0 and then filtered using Hyflo super-cell as a filter aid. The mycelium is discarded and the filtrate is extracted with an amount of butanol corresponding to about 1/2 of its volume. The organic phase is separated from the aqueous one, and, after washing with acidic water (pH 4.0) is concentrated to about 1/10 of its original volume and allowed to stand for 10-12 hours at a temperature of 3°-6° C. A crude precipitate forms, which is collected on filter, washed with butanol and dried under vacuum at room temperature: yield 3.0 g. Chromatographic assays on Whatman paper No. 1 or on silica-gel of this crude precipitate, and subsequent microbiological development of the spots by using Staphylococcus aureus as the detecting system, indicate the presence of two components which are defined as metabolite A and metabolite B (gardimycin): they have different R_(f) values which depend on the nature of the employed eluting system.

The crude mixture is further purified by dissolving in about 30 ml. of water. The resulting solution is dialyzed for about 16 hours against distilled water and then concentrated to small volume under vacuum. 1.5 Grams of rough antibiotic substance are obtained, which still is a mixture of metabolite A and gardimycin. The two antibiotic substances are separated and purified by several countercurrent extractions, by relying upon the different partition coefficients of component A and gardimycin in the predetermined solvent system. The employed solvent system consists of butanol:sodium-potassiumphosphate buffer M/15 pH 7.2: hexane in the ratio 1:1:0.1; the partition coefficients in this medium of metabolite A and gardimycin are 0.3 and 0.8 respectively. After 100 extractions, 0.450 g. of gardimycin as its monosodium salt are obtained.

The mother liquors from the butanol extract after precipitation of the mixture of metabolites A and B are further concentrated to small volume and then poured into an excess of light petroleum. A precipitate readily forms, which is recovered by filtration. Chromatographic assays carried out under the same conditions as before indicate that this fraction consists of one product which has different R_(f) value from metabolites A and B in the same eluting systems. This fraction is named metabolite C.

The different chromatographic-patterns of the three metabolites in the various eluting systems are reported in the following table:

    __________________________________________________________________________     Chromatographic patterns of metabolite A, metabolite B (gardimycin) and        metabolite C.                                                                  Chromatography on Whatman paper No. 1                                          Visualization of the spots by microbiological development                                                    R.sub.f values                                   on Staphylococcus aureus              Metabolite B                             ELUTING SYSTEM                Metabolite A                                                                           (gardimycin)*                                                                          Metabolite                       __________________________________________________________________________                                                   C                                1)                                                                               Butanol saturated with phosphate buffer M/15 pH 6.0                                                        0.00    0.15    0.85                             2)                                                                               Butanol saturated with water containing 2% of                                  p-toluenesulfonic acid      0.75    0.80    0.95                             3)                                                                               Butanol saturated with water containing 2% of                                  ammonium hydroxide          0.15    0.10    0.85                             4)                                                                               Phosphate buffer M/15 pH. 6.0 saturated with butanol                                                       0.65    0.80    0.00                             5)                                                                               20% aqueous solution of sodium chloride                                                                    0.00    0.80    0.00                             6)                                                                               Butanol:methanol:water=40:10:20 with 0.75% of methyl                           orange                      0.70    0.65    0.90                             7)                                                                               Butanol:methanol:water=40:10:20                                                                            0.85    0.60    0.90                             8)                                                                               Acetone:water=1:1           0.80    0.80    0.65                             9)                                                                               Ethyl acetate saturated with water                                                                         0.00    0.00    0.75                             Thin-layer chromatography on silicagel.                                        Visualization of the spots by sulfuric acid and vanilline                      and microbiological development on Staphylococcus aureus                       ELUTING SYSTEM                                                                 Ammonium hydroxide:ethanol:water=1:8:1                                                                       0.30    0.70    0.00                             __________________________________________________________________________      *Metabolite B is tested as the monosodium salt                           

EXAMPLE 2

By operating as described in Example 1 the strain Actinoplanes Liguriae ATCC 31048 is aerobically cultivated for 130-170 hours at about 28°-30° C. 1.4 Grams of antibiotic activity are obtained, which are a mixture of metabolite A and metabolite B (gardimycin). The existence of the two metabolites has been detected by means of the same chromatographic assays reported in Example 1. The purification of the obtained antibiotic activity and the separation of the two metabolites has been achieved as in Example 1. 0.2 Grams of gardimycin as the monosodium salt are obtained.

Chemico-physical properties of gardimycin as the monosodium salt

Gardimycin (as the monosodium salt) is an amorphus white powder with amphoteric character. It has an isoelectric point of 4.2 which was determined by electrofocalization. Upon strong hydrolysis in hydrochloric acid 6N at 120° in a closed funnel for 16 hours and chromatographic analysis of the hydrolysis products it was possible to evidence the following amino-acids; valine, serine, glycine, glutamic acid, isoleucine, leucine and alanine. After alkaline hydrolysis with barium hydroxide at 110° in a closed funnel for 15 hours and chromatographic analysis of the hydrolysis products it is possible to evidence the presence of tryptophane. The above mentioned amino-acids are present in the following ratio:

    ______________________________________                                         Amino-acids      Approximate ratio                                             ______________________________________                                         Valine           2                                                             Serine           1                                                             Glycine          2                                                             Glutamic acid    1                                                             Isoleucine       2                                                             Leucine          1                                                             Alanine          1                                                             Tryptophan       1                                                             ______________________________________                                    

Moreover, the analysis of the products resulting from the acidic hydrolysis reveals the presence of further amino-acids of undetermined structure, some of them containing sulfur. Furthermore, gardimycin (as the monosodium salt) is characterized by the following properties:

(1) Melting point: 260° C. (decomposition).

(2) Molecular weight: 2005-2168.

(3) Elemental analysis: C = 48.7-48.6%; H = 6.7-6.6%; N = 11.8-12.2%; S = 5.3-5.5%; Na = 1.1%; H₂ O = 3.6-3.3%.

(4) U.V. absorpion bands: In each of the below outlined solvent systems, gardimycin shows the following values:

    ______________________________________                                                         λ (mμ)                                               Solvent         max           E.sub.1.sup.1% cm.                               ______________________________________                                         methanol        273 (shoulder)                                                                 280           26                                                               299           24                                               sodium hydroxide 0.1N                                                                          273 (shoulder)                                                                 279           26                                                               288           22                                               hydrochloric acid 0.1N                                                                         273 (shoulder)                                                                 279           26                                                               288           22                                               phosphate buffer                                                                               273 (shoulder)                                                 pH. 7.38        279           24                                                               288           20                                               ______________________________________                                    

The complete picture of the spectrum is given in FIG. 1.

(5) Infrared spectrum: Characteristic absorption bands in nujol have been observed at the following frequencies: (cm⁻ 1): 3280, 2920-2840 (nujol); 1650, 1520, 1455 (nujol), 1375 (nujol), 1260, 1045, 990, 720.

The complete picture of the spectrum is given in FIG. 2.

(6) Specific rotation: [α]_(D) ²⁵ = -44° (c = 0.5% dimethylformamide).

(7) Solubility: soluble in water, aqueous sodium bicarbonate, diluted aqueous solutions of alkali hydroxides, hot methanol, dimethylformamide, dimethylsulfoxide, glacial-acetic acid. Insoluble in diluted mineral acids, benzene, acetone, chloroform, carbon tetrachloride, (C₂₋₆)-aliphatic alkanols, tetrahydrofuran.

(8) Characteristic reactions:

    ______________________________________                                         Fehling            positive                                                    Tollens            positive                                                    KMnO.sub.4         positive                                                    H.sub.2 SO.sub.4.sup.4 conc.                                                                      positive                                                    Ninhydrin          negative                                                    FeCl.sub.3         negative                                                    Millon             negative                                                    Schiff             negative                                                    Maltol             negative                                                    ______________________________________                                    

(9) Ionizable functions: An ionizable function is potentiometrically evidenced with pK_(a) =7.1 (water) and pK_(a) =8.5 (methylcellosolve:water: 16:4).

(10) Furthermore, starting from the monosodium salt of gardimycin it is possible to prepare the following derivatives:

(a) Gardimycin free acid: 1.0 g of gardimycin monosodium salt is dissolved in 150 ml. of water. The resulting solution is brought to pH 2.5 by adding aqueous 10% hydrochloric acid and is then extracted two times with 75 ml. of butanol saturated with water. The butanol extracts are collected and concentrated in vacuo at 45° C. to a volume corresponding to 1/20 of the initial volume. After standing at 4° C. for 12 hours a precipitate forms, which is collected, washed with light petroleum and dried in vacuo at 40°-45° C. 0.950 Grams of product are obtained which decomposes between about 250° and about 300° C.

(b) Gardimycin disodium salt: 1.0 g. of gardimycin monosodium salt is dissolved in 150 ml. of water and the pH of the resulting solution is brought to 9.6 by adding N/10 sodium hydroxide. The solution is then lyophilized. 0.900 Grams of product are obtained melting at 250° C.

(c) Gardimycin calcium salt: 0.3 g. of gardimycin monosodium salt are dissolved in 20 ml. of water and the resulting solution is slowly added, under stirring, with several portions of a saturated solution of calcium chloride until a complete precipitation is reached. The obtained solid is filtered, dried in vacuo and dissolved in 10 ml. of acetone. After pouring into 200 ml. of diethylether a precipitate forms, which is filtered and dried in vacuo at 40°-45° C. 0.2 Grams of product are obtained which decomposes at 330°-40° C. 

We claim:
 1. A process for producing Gardimycin which comprises cultivating a strain selected from the group consisting of Actinoplanes garbadinensis ATTCC 31049 and Actinoplanes liguriae ATCC 31048 under aerobic conditions, in an aqueous nutrient medium containing an assimilable source of carbon, an assimilable source of nitrogen and inorganic salts, until a substantial antibiotic activity is present in the medium, recovering said antibiotic activity therefrom, and separating it from the medium.
 2. A process as defined in claim 1, wherein the fermentation is carried out at a temperature between about 25° and about 35° C.
 3. A process as defined in claim 1, wherein the fermentation is carried out for a time ranging from about 96 to 170 hours.
 4. The antibiotic substance gardimycin having the following characteristics (as the monosodium salt):1. Melting point: 260° C (decomposition);
 2. Molecular weight: 2005-2168;
 3. Elemental analysis: C=48.7-48.6%; H=6.7-6.6% N=11.8-12.2%; S=5.3-5.5%; Na=1.1%; H₂ O=3.6-3.3%;
 4. u.v. absorption bands: In each of the below outlined solvent systems, gardimycin shows the following values:

    ______________________________________                                                         λ(m)                                                    Solvent         max           E.sub.1.sup.1% cm.                               ______________________________________                                         methanol        273 (shoulder)                                                                 280           26                                                               299           24                                               sodium hydroxide 0.1N                                                                          273 (shoulder)                                                                 279           26                                                               288           22                                               hydrochloric acid 0.1N                                                                         273 (shoulder)                                                                 279           26                                                               288           22                                               phosphate buffer                                                                               273 (shoulder)                                                 pH. 7.38        279           24                                                               288           20                                               ______________________________________                                    

The complete picture of the spectrum is given in FIG. 1;
 5. 5. infrared spectrum: characteristic absorption bands in nujol have been observed at the following frequencies (cm⁻ ¹): 3280, 2920-2840 (nujol), 1650, 1520, 1455 (nujol), 1375 (nujol), 1260, 1045, 990, 720;The complete picture of the spectrum is given in FIG. 2;
 6. specific rotation: [α] = -44° (c = 0.58% dimethylformamide);
 7. Solubility: soluble in water, aqueous sodium bicarbonate, diluted aqueous solutions of alkali hydroxides, hot methanol, dimethylformamide, dimethylsulfoxide, glacial-acetic acid. Insoluble in diluted mineral acids, benzene, acetone, chloroform, carbon tetrachloride, (C₂₋₆)-aliphatic alkanols, tetrahydrofuran;
 8. Characteristic reactions:

    ______________________________________                                         Fehling            positive                                                    Tollens            positive                                                    KMnO.sub.4         positive                                                    H.sub.2 SO.sub.4 conc.                                                                            positive                                                    Ninhydrin          negative                                                    FeCl.sub.3         negative                                                    Million            negative                                                    Schiff             negative                                                    Maltol             negative                                                    ______________________________________                                    


9. Ionizale functions: an ionizable function is potentiometrically evidenced with pK_(a) =7.1 (water) and pK_(a) =8.5 (methylcellosolve:water= 16:4);
 10. Isoelectric point: 4.2 (determined by electrofocalization);
 11. R_(f) values:

    ______________________________________                                         Chromatography on Whatman paper No. 1                                                                     R.sub.f values                                      Visualization of the spots by micro-                                           biological development on Staphyloccus                                         aureus                                                                         ELUTING SYSTEM                                                                 1)   Butanol saturated with phosphate buffer                                                                  0.15                                                 M/15 pH 6.0                                                               2)   Butanol saturated with water containing                                        2% of p-toluenesulfonic acid                                                                             0.80                                            3)   Butanol saturated with water containing                                        2% of ammonium hydroxide  0.10                                            4)   Phosphate buffer M/15 pH 6.0 saturated                                         with butanol              0.80                                            5)   20% aqueous solution of sodium chloride                                                                  0.80                                            6)   Butanol:methanol:water=40:10:20 with                                           0.75% of methyl orange    0.65                                            7)   Butanol:methanol:water=40:10:20                                                                          0.60                                            8)   Acetone:water=1:1         0.80                                            9)   Ethyl acetate saturated with water                                                                       0.00                                            Thin-layer chromatography on silicagel.                                        Visualization of the spots by sulfuricacid                                     and vanilline and microbiological develop-                                     ment on Staphylococcus aureus                                                  ELUTING SYSTEM                                                                 Ammonium hydroxide:ethanol-water=1:8:1                                         ______________________________________                                    

The antibiotic being further characterized by the following approximate ratio of amino-acids of identified structure:

    ______________________________________                                         Amino-acid       Approximate ratio                                             ______________________________________                                         Valine           2                                                             Serine           1                                                             Glycine          2                                                             Glutamic acid    1                                                             Isoleucine       2                                                             Leucine          1                                                             Alanine          1                                                             Tryptophan       1                                                             ______________________________________                                    


5. The antibiotic substance gardimycin a defined in claim 4 as the free acid.
 6. The antibiotic substance gardimycin as defined in claim 4 as the disodium salt.
 7. The antibiotic substance gardimycin as defined in claim 4 as the calcium salt. 